U.S. patent application number 10/494277 was filed with the patent office on 2005-03-10 for use of chitosan materials.
Invention is credited to Hahnemann, Birger, Lotzbeyer, Thomas, Pahmeier, Andrea, Sperling, Philipp.
Application Number | 20050054610 10/494277 |
Document ID | / |
Family ID | 26010481 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054610 |
Kind Code |
A1 |
Hahnemann, Birger ; et
al. |
March 10, 2005 |
Use of chitosan materials
Abstract
The invention relates to the use of a biocompatible material
based on chitosan and an acid, e.g. as flexible film or/and porous
matrix, as remedy in the area of neurosurgery, in particular as
nerve splint, and for repairing tendons and ligaments.
Inventors: |
Hahnemann, Birger; (Zossen,
DE) ; Pahmeier, Andrea; (Zossen, DE) ;
Sperling, Philipp; (Berlin, DE) ; Lotzbeyer,
Thomas; (Munchen, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
26010481 |
Appl. No.: |
10/494277 |
Filed: |
October 19, 2004 |
PCT Filed: |
October 30, 2002 |
PCT NO: |
PCT/EP02/12112 |
Current U.S.
Class: |
514/55 |
Current CPC
Class: |
A61L 31/042 20130101;
A61L 27/20 20130101; A61L 27/20 20130101; A61L 31/042 20130101;
C08L 5/08 20130101; C08L 5/08 20130101; A61L 2430/32 20130101 |
Class at
Publication: |
514/055 |
International
Class: |
A61K 031/722 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 2001 |
DE |
101 53 470.1 |
Oct 8, 2002 |
DE |
102 46 816.8 |
Claims
1. The use of a biocompatible material based on chitosan and an
acid as remedy in the area of neurosurgery.
2. The use as claimed in claim 1 for extracorporeal or
intracorporeal nerve reconstruction.
3. The use as claimed in claim 1, characterized in that the
material is in the form of a flexible, in particular nonporous,
film.
4. The use as claimed in claim 3, characterized in that a film with
a preferred direction of curling is employed.
5. The use as claimed in claim 3 as nerve splint for wrapping round
nerves.
6. The use as claimed in claim 2 for growing Schwann cells on the
film.
7. The use as claimed in claim 1, characterized in that the
material is in the form of a porous matrix.
8. The use as claimed in claim 7 as neurological swab.
9. The use as claimed in claim 7 as matrix for uniting nerve
ends.
10. The use as claimed in claim 9 for the ingrowth of neurons into
the porous matrix.
11. The use as claimed in claim 1, characterized in that the
material is in the form of a combination of a flexible film and of
a porous matriz.
12. The use as claimed in claim 11, characterized in that film and
matrix are employed as separate components.
13. The use as claimed in claim 11, characterized in that film and
matrix are employed as composite component.
14. The use as claimed in claim 1 for producing a neuron
microprobe.
15. The use as claimed in claim 1 for producing a neuron
microprobe.
16. The use as claimed in claim 15 for anchoring tendons or
ligaments to bone.
17. The use of a biocompatible material based on chitosan and an
acid as swab or tampon for abscess cavities.
18. The use of a biocompatible material based on chitosan and an
acid for covering tissue and organs after injuries and/or during
surgical procedures.
19. A biocompatible material based on chitosan and an acid, that
film and matrix are employed as composite component, characterized
in that it is in the form of a film with a preferred direction of
curling.
20. A film as claimed in claim 19, characterized in that it has a
thickness in the range from 1 .mu.m to 100 .mu.m.
Description
[0001] The invention relates to the use of a biocompatible material
based on chitosan and an acid, e.g. as flexible film or/and porous
matrix, as remedy in the area of neurosurgery, in particular as
nerve splint, and for repairing tendons and ligaments.
[0002] German patent application 199 48 120.2 discloses a method
for producing a biocompatible three-dimensional matrix, wherein an
aqueous solution of a chitosan and of an acid, which is present in
excess, in particular a hydroxy carboxylic acid, is frozen, and the
water is removed by sublimation under reduced pressure, with the
excess acid being removed, in particular neutralized, before the
freezing or after the removal of the water by sublimation. A matrix
which can be obtained by the method and which can be used for
producing implants is also disclosed.
[0003] German patent application 101 17 234.6 discloses
biocompatible nonporous materials based on chitosan and an acid, in
particular a hydroxy carboxylic acid. These materials may for
example be in the form of a film.
[0004] Based on this knowledge, it was the object of the present
invention to provide novel applications for materials based on
chitosan and an acid, in particular a hydroxy carboxylic acid.
[0005] A first aspect of the present invention therefore relates to
the use of a biocompatible material based on chitosan and an acid,
in particular a hydroxy carboxylic acid, as remedy in the area of
neurosurgery, for example for extracorporeal or intracorporeal
nerve reconstruction.
[0006] In a first embodiment, the material is in the form of a
flexible, in particular nonporous, film. The film has a thickness
of, preferably, 1 .mu.m-200 .mu.m, particularly preferably of 10
.mu.m-50 .mu.m and is obtainable by:
[0007] providing an aqueous solution of a chitosan and of an acid,
in particular a hydroxy carboxylic acid, which is present in
excess,
[0008] drying the solution without freezing and
[0009] removing excess acids before or/and after drying, preferably
by neutralization.
[0010] The film can be produced in prefabricated strips with a
width of, for example, 1 mm-10 mm. Alternatively, the required
pieces of film can also be made as required, e.g. during an
operation.
[0011] In a particularly preferred embodiment there is use of a
film with a memory effect, i.e. with a preferred direction of
curling. This means that the film tends, under the conditions of
use, to curl or form rolls. The film with memory effect can be
produced in a simple manner by removing the acid, e.g.
neutralizing, from one side of the film article
[0012] The film, in particular the film with memory effect, is
suitable as nerve splint for wrapping round nerves. It has been
found that, in particular, Schwann cells are able to grow well on
the film. The nerve splint can also be employed for example in
traumatology for initial management of transected nerve ends and in
reconstructive surgery.
[0013] A further area of use of the film is for wrapping round
tendons and ligaments, in which case it is beneficial to use a
rolled film. It has been found that the union of severed tendons
and ligaments is promoted by tension-free wrapping with a film of
the invention. The enveloping can be adapted to the dimensions of
the wrapped ligaments and tendons. Under these conditions,
tenocytes, e.g. including human tenocytes, show a distinctly better
proliferation than in other matrices.
[0014] The film can be used as carrier for a porous
three-dimensional matrix. It is thus possible to provide
biocompatible composite materials which include at least one
biocompatible film as described above, and at least one
biocompatible porous matrix. The biocompatible porous matrix is
preferably based on chitosan and an acid, in particular a hydroxy
carboxylic acid. However, it is also possible to use other porous
biocompatible matrices.
[0015] In a further preferred embodiment, the biocompatible
material based on chitosan and an acid is a porous matrix. It is
particularly preferred to use a biocompatible porous matrix as
disclosed in the German application 199 48 120.2, which is
obtainable by:
[0016] providing an aqueous solution of a chitosan and of an acid,
in particular a hydroxy carboxylic acid, which is present in
excess,
[0017] freezing and drying the solution, in particular by
sublimation under reduced pressure and
[0018] removing excess acid before or/and after the freezing, in
particular by neutralization with a suitable base, e.g. NaOH.
[0019] The porous matrix can be employed for example as swab or/and
tampon for medical or veterinary medical applications, e.g. as
neurological swab, as matrix for uniting nerve ends and/or as
tampon for abscess cavities. It has been found that the porous
matrix is particularly suitable for the ingrowth of neurons. A
further advantage of the porous matrix is the high swelling
capacity to 10 times the initial weight or more.
[0020] The porous matrix can be produced in prefabricated pieces
with a volume of, for example, 1-10 mm.sup.3. Alternatively, the
pieces required can be made as required, e.g. during an
operation.
[0021] In a further embodiment, the material can be in the form of
a combination of a flexible film and of a porous matrix. The film
and the matrix can in this case be employed as separate components,
for example for reconstructing severed nerves. An example of such a
combination of materials, in which the film and matrix are employed
as separate components, is shown in FIGS. 1A, 1B and 1C. As shown
in FIG. 1A, a porous matrix (6) is inserted between the ends (2, 4)
of a severed nerve fiber. A film (8) is wound in one or more turns
around the nerve filament. The film (8) can be fixed with a
suitable adhesive, e.g. a fibrin glue or a tissue glue, for
fixation to the nerve. FIG. 1B shows a cross section through the
matrix (6) and the film (8), with the film being wound more than
once. In FIG. 1C there is only one complete winding of the film (8)
around the matrix (6).
[0022] Tendons and ligaments can be repaired in a manner analogous
to that shown in FIG. 1. In addition, severed tendons and ligaments
can also be anchored to bone. For this purpose, the bone, into
which a depression can be cut where appropriate, is covered by a
porous matrix into which tenocytes of the porous tendon or of the
ligament can grow, where appropriate after wrapping in a rolled
film, bringing about stable anchoring to the bone.
[0023] Yet a further preferred application of the film relates to a
use as biohybrid implant, e.g. as capsular or tubular structure
where appropriate in combination with the porous matrix for
encapsulating cells, especially cells which can be electrically
stimulated. In one embodiment, the implant is a neuron microprobe.
In this case there is provision of an envelope consisting of the
film, e.g. in the form of a bag or of a tubular structure, into
which neuronal cells, which have been genetically manipulated where
appropriate, are introduced. The envelope is implanted in the body
and may serve, where appropriate after electrical stimulation to
regenerate nerves, e.g. peripheral nerves, as pain pump (Erb et
al., Exp. Neurol. 124, (1993), 372-376).
[0024] Moreover, the film can also serve as pain pump, where
appropriate in combination with the matrix. This pain pump is an
implant which has through external stimuli a controllable release
of endorphins/enkephalins in the brain/subarachnoid space for the
purpose of treating very severe chronic states of pain, as is the
case for example in spinal disorders and tumor diseases.
[0025] In order to achieve genetically modified cells and the
electrical stimulation, the film is used to sew a type of bag in
which the biohybrid implant is located. Besides the film as
external sheath, the matrix can additionally serve as carrier for
the chromaffin cells which later release analgesic peptides in
response to the electrical stimuli.
[0026] The chitosan-based material, especially the film, can
additionally serve as covering for tissues and organs, e.g. the
brain after injuries and/or during surgical procedures.
[0027] In an alternative embodiment it is also possible for film
and matrix to be employed as composite components, with film and
matrix each being disposed alternately in layers. Examples of such
multilayer systems are depicted in FIGS. 2A, 2B and 2C. An
alternative possibility is also to dispose a nonporous film between
two porous matrices.
[0028] The nonporous film of the invention, the porous matrix or
the composite system based thereon can also be used for the in
vitro cultivation of neuronal cells. In this case, the materials
may comprise additional factors for cell growth, e.g.
cytokines.
[0029] The porous matrix may where appropriate have anisotropic
structures, for example fibers or/and chambers in parallel
alignment. The anisotropic matrix is obtainable by:
[0030] providing an aqueous solution of a chitosan and of an acid,
in particular a hydroxy carboxylic acid, which is present in
excess,
[0031] anisotropic freezing and drying of the solution, in
particular by sublimation under reduced pressure and
[0032] removing excess acid before or/and after the freezing.
[0033] The anisotropic freezing preferably comprises a freezing
with use of structured cooling elements, e.g. tubes in direct or
indirect contact with the matrix during the freezing process. The
cooling elements may be elongate in order, for example, to obtain
fibers or chambers in parallel alignment in the matrix. However,
curved structures, e.g. simulations of the organ to be shaped, can
also be used as cooling elements.
[0034] The anisotropic porous matrix can be employed in a
biocompatible composite material system together with another
material, for example with a biocompatible nonporous film. The
anisotropic matrix, or the composite material system based thereon,
can be employed for the in vitro cultivation of cells or as implant
without previous cell colonization, corresponding to the
applications mentioned above.
[0035] The matrices and films of the invention based on chitosan
and acids are produced substantially by the method indicated in the
German applications 199 48 120.2 and 101 17 234.6, unless indicated
otherwise. Preferably, firstly an aqueous solution of a partially
deacetylated chitosan and of an acid, which is present in excess,
is prepared. Excess means in this connection that the aqueous
solution has an acidic pH, preferably below pH.ltoreq.4. The free
amino groups of the chitosan are at least in part protonated
thereby, thus increasing the solubility in water. The amount of
acid is not critical. It must merely be chosen so that the chitosan
dissolves. Excessive addition of acid is avoided where possible,
because excess acid must be removed again, and the working up is
made difficult thereby when the amounts of acid are large.
Favorable amounts of acid are those yielding a 0.05 to 1 N,
preferably 0.1 to 0.5 N, in particular 0.1 to 0.3 N, solution. The
amount of chitosan is preferably chosen to result in a 0.01 to 0.5
M, preferably 0.1 to 0.3 M, solution. The structure of the matrix,
in particular the pore size thereof, can be influenced by the
concentration of the chitosan solution. It is possible in this way
to adapt the pore size of the matrix to the particular cell type
with which the matrix is to be colonized.
[0036] Because chitosan is produced from natural sources it has no
uniform molecular weight. The molecular weight may be between 20
kDa to more than 1000 kDa, depending on the source and method of
processing.
[0037] The chitosan for producing the three-dimensional matrix is
not subject to any restrictions in relation to its molecular
weight. The aqueous chitosan solution is prepared by using an acid
which is an inorganic acid or, preferably, an organic acid,
particularly preferably an alkyl or aryl hydroxy carboxylic acid.
Hydroxy carboxylic acids having 2 to 12 carbon atoms are
particularly suitable, it being possible for one or more hydroxyl
groups and one or more carboxyl groups to be present in the
molecule. Specific examples are glycolic acid, lactic acid, malic
acid, tartaric acid, citric acid and mandelic acid. Lactic acid is
particularly preferred.
[0038] In producing a porous matrix, the solution of chitosan and
acid is initially at least partially neutralized by adding base and
then frozen or directly frozen without previous neutralization.
Neutralization before freezing is preferred. The pH after the
neutralization is generally 5.0 to 7.5, preferably 5.5 to 7.0 and
in particular 6.0 to 7.0.
[0039] After the freezing, the water is removed by sublimation
under reduced pressure, for example in the pressure range from
0.001 to 3 hPa.
[0040] To produce a nonporous film, the solution is not subjected
to freezing and sublimation, but is dried without freezing at
optionally elevated temperature or/and reduced pressure, and is
preferably neutralized after drying. The resulting nonporous matrix
has a high load-bearing capacity and extensibility in the moist
state.
[0041] The large number of amino and hydroxyl groups makes the
material modifiable as desired. In a preferred embodiment, ligands
are covalently or noncovalently bound to the chitosan, preferably
to the free amino groups of the chitosan. Ligands which can be used
are, for example, growth factors, proteins, hormones, heparin,
heparan sulfates, chondroit sulfates, dextran sulfates or a mixture
of these substances. The ligands preferably serve to control and
improve cell proliferation.
[0042] Cell growth on the matrix or the film is further improved if
the matrix is coated with autologous fibrin.
[0043] The three-dimensional matrix can be colonized both by human
and by animal cells (for example from horse, dog or shark). Shark
cells are particularly suitable because they induce a negligible
immunological response in the recipient.
[0044] The materials as described above can be employed in the
human medical and veterinary sectors. Further areas of application
are the use as disposable article, for example as swab.
[0045] The materials are sterilized before use in the cell culture,
in order to guarantee freedom from germs. The sterilization can
take place by thermal treatment, e.g. by autoclaving, steam
treatment etc. or/and by irradiation, e.g. gamma-ray treatment. The
sterilization preferably takes place in a physiologically tolerated
buffered solution, e.g. in PBS, in order to ensure thorough wetting
of the matrix or the film with liquid and the absence of major air
inclusions.
[0046] When the cells are cultured, the material is degraded within
a period of about 5-8 weeks or longer. The degradation times can be
adjusted via the degree of deacetylation of the chitosan and the
concentration of the material.
[0047] The invention is further to be explained by the following
figures and examples.
[0048] Explanation of the Figures:
[0049] Illustration 1 shows in FIGS. 1A, 1B and 1C combinations of
materials in which film and matrix are employed as separate
combinations.
[0050] Illustration 2 shows in FIGS. 2A, 2B and 2C alternative
embodiments.
[0051] Illustration 3: pictures A and B: Schwann cells (rat adult,
10% FCS), uncoated; pictures C and D: PORN/laminin, pictures E and
F: PLL; plane of focus: culture dish (A, C, E); film* (B, D,
E).
[0052] Illustration 4: rat spinal ganglionic neuron cultures (P1),
PORN-laminin coated plastic (left); uncoated film (right), 24 h in
culture.
[0053] Illustration 5: spinal ganglionic neurons (diss. P1, rat)
under serum-free conditions (+NGF), 3 days of culturing. Some
neurons become detached from the matrix during the histological
workup. Nevertheless, differentiated neurons with axons are found
in association with the matrix (see arrows in C, E, F).
[0054] Illustration 6: Axonal regeneration (rat, adult, sciatic
nerve) 8 weeks after implantation of a "wound" film graft. The
wound film is incorporated in the connective tissue. The nature of
the incorporation (no cells in the cavities) suggests that the
"fraying" on the inside of the film (A*, D*) is not attributable to
postoperative enzymatic synthesis. Regenerating axons in some cases
grow as large fascicles into the individual lamellae (proximal
junction, see B).
[0055] Illustration 7: axional regeneration (rat, adult, sciatic
nerve) of three experimental animals (A/B; C/D; E/F) 8 weeks after
implantation of a "wound" film graft. Survey magnifications
(4.times., A, C, E) of the proximal nerve-film junction, and
detailed magnifications of the distal nerve stump (10.times.) with
regenerated axons (see arrow).
EXAMPLE 1
Production of a Nonporous Film
[0056] A mixture of chitosan and lactic acid is prepared by the
method described in Example 3 of DE 199 48 120.2. The solution is
poured into a Petri dish, dried at 50.degree. C. and, after a
glass-clear film has formed, neutralized with 1 M sodium hydroxide
solution to a pH of 7. The resulting film has a high load-bearing
capacity and extensibility in the moist state.
[0057] A film with memory effect can be generated by specific
addition of the sodium hydroxide solution onto one side.
EXAMPLE 2
In Vitro Cultivation of Schwann Cells and Neurons
[0058] Schwann cells and spinal ganglionic neurons were put onto
the film or the matrix and cultured in vitro. The film is
particularly suitable for culturing Schwann cells thereon
(Illustration 3 and 4).
[0059] Neurons are successfully cultured in the matrix especially
when the pore diameters are about 10-20 .mu.m (Illustration 5).
EXAMPLE 3
Atraumatic Nerve Approximation Using a Film with Memory Effect
[0060] Principle and Surgical Method:
[0061] Two nerve stumps are connected by means of the self-curling
chitosan film, and the ends are fixed using commercially available
fibrin glue. The film is spread using forceps, and the nerve stumps
are placed thereon. After removal of the forceps, the film curls up
of its own accord (memory effect) and encloses the nerve ends.
[0062] Advantages of the Method:
[0063] No microsurgical suture is necessary, i.e. the procedure can
be performed easily even by a clinician with no microsurgical
experience, i.e. even by a traumatologist.
[0064] The curling up of the film avoids a pressure on the nerve
ends: in the event of swelling, which regularly occurs after
severance of a nerve, of the nerve stumps, the film is easily able
to adapt to the increased diameter without exerting a pressure
effect on the nerve ends. This avoids a substantial disadvantage of
artificial nerve grafts customary at present, namely the secondary
nerve damage from circular structures of constant diameter.
[0065] Results:
[0066] A total of 10 nerves was investigated after implantation in
the rat model for eight weeks (Illustrations 6 and 7).
[0067] Results:
[0068] 1. Ingrowth of regenerating nerve fibers into the grafts
took place in all the animals investigated. The fibers grew between
the lamellae of the curled up film.
[0069] 2. The width of the grafts led to multiple curling. A single
curling up would be ideal, so that the end result is a tube with
only one slit.
[0070] 3. The distal nerve stump was reached in all the animals
investigated.
[0071] Conclusions from the In Vivo Experiments:
[0072] A nerve splinting using a chitosan film with memory effect
is possible. The nerve splinting allows nerve approximation even if
a dehiscence is present between the nerve ends. At present, this
still requires implantation of a nerve graft, e.g. from a cutaneous
nerve of the leg. Coating with film with Schwann cells can achieve
an increased rate of regeneration.
* * * * *